Morphological Character Mapping on a Molecular Phylogeny Using Pollen Variation in the Cryptanthinae (Boraginaceae)

Home , Acanthocarpus (plant), Amsinckia, Amsinckia menziesii, Amsinckia spectabilis, Chaetolepis, Cryptantha

Rachel Spaeth

A thesis submitted to Sonoma State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE in

Biology

______Dr. Richard Whitkus, Chair

______Dr. Michelle Goman

______Dr. Murali Pillai

______Date

Authorization for Reproduction of Master’s Thesis

Permission to reproduce this thesis in part or entirety must be obtained from me.

DATE:______Signature

______Street Address

______City, State, Zip

iii

Morphological Character Mapping on a Molecular Phylogeny: Using Pollen Variation in the Cryptanthinae (Boraginaceae)

Thesis by Rachel Spaeth

ABSTRACT

Phylogenetic classification at the species level in the Boraginaceae is notoriously difficult when relying solely on morphological data. Studies are currently in progress to generate a well-supported phylogenetic tree of this family using molecular data. A molecular based phylogeny may reveal the characters that evolved slowly enough to have the same state in closely related taxa found in some key palynological traits used in previous classifications. Pollen attributes were collected on seventy four species across six genera in the subtribe Cryptanthinae using Scanning Electron Microscopy (SEM). The pollen data exhibit features which are taxonomically informative including shape, aperture type, sculpturing, and size. Cryptanthinae pollen encompasses three of the nine Erdtman (1966) shape categories, and seven of the eleven Faegri and Iversen (1975) subshape categories. Their aperture types include heterocolpate, zonoporate, and zonocolpate forms. They are sculpted with fossulate, foveolate, echinate, reticulate, and gemmate clavate surfaces. They range in size from 4.85μm long and 1.92μm wide to 40.85μm long and 25.60μm wide. Some of them have a transverse groove and others do not. The same is true for the presence or absence of polar apertures. These characteristics were mapped on a molecular phylogeny to observe evolutionary trends. Biogeographic data such as habitat moisture, range of distribution, flowering period, and style type were also mapped on the molecular phylogeny to uncover selection pressures responsible for the high level of morphological diversity in this subfamily. This analysis revealed habitat moisture level as one of the driving forces behind pollen subshape diversity in the Cryptanthinae.

Chair: ______Signature

MS Program: Biology

Sonoma State University Date: ______

Acknowledgement

Funding provided by the California Rare Fruit Growers Association Redwood

Empire Chapter and the California Native Plant Society Milo Baker Chapter. Special thanks to Dr. Richard Whitkus, Matthew Guilliams, Steve Anderson, Dr. Murali Pillai,

Dr. Michelle Goman, Daniel Streeter, The William M. Keck Microanalysis Laboratory, the Northcoast Herbarium, the University of California at Berkeley / Jepson Herbarium,

Universidad de Conceptión Herbario, and Missouri Botanical Garden Herbarium.

Table of Contents

Introduction…………………………………..……………………..…...………………... 1

Research Questions………………………………………………..…..………………...... 9

Methods…………………………………………………………………..………………..9

Results……………………………………………………….…………………………... 15

Morphological Variation………………………………………....……...... 15

Pollen Size………………………………………………………………………... 15

Pollen Shape…………………………………………………………………….... 17

Pollen Subshape……………………………………….…………………………..18

Pollen Apertures……………………………………….….……………………… 20

Pollen Sculpturing…………………………………………………...... 21

Phylogenetic Trait Distribution………………………………….……...... 22

Pollen Size………………………………………………...……………………… 24

Pollen Shape………………………………………………...... 24

Pollen Subshape…………………………………………...... 26

Pollen Apertures………………………………….……….……………………….28

Pollen Sculpturing………………………………………....……………………... 30

Habitat, Biogeography, and Breeding System………………….……...... 32

Habitat Moisture…………………………………………………..……………… 32

Range…………………………………....…………….....…………...... 34

Flowering Period…………………………………………...... 36

Breeding System……………………………………………………...... 36

Biogeographic Correlation with Morphological Features……………………………. 36

Discussion……………………………………………………………...……………….. .38

Cryptanthinae and Geologic Time…………………………………………………… 38

Noteworthy Cryptanthinae Clades…………………………………………………… 39

Conclusion………………………………………………….…………...………………. 44

Literature Cited…………………………………………………………...... 45

Appendix 1………………………………………………….…………..……………….. 51

Appendix 2…………………………………………………………..….……………….. 54

Appendix 3…………………………………………………………...………………….. 67

Appendix 4……………………………………………………..…….………………….. 80

Appendix 5………………………………………………………………...... 81

Appendix 6……………………………………………….………………...... 81

Appendix 7………………………………………………………………………………. 82

Appendix 8………………………………………………………………………………. 82

Appendix 9………………………………………………………………………………. 83

Appendix 10……………………………………………………………………………... 83

Appendix 11……………………………………………………………………………... 84

Appendix 12……………………………………………………………………………... 86

vii

List of Tables

Table 1. Pollen size variation observed across genera in the subtribe Cryptanthinae (Boraginaceae). Polar (P) and equatorial (E) lengths were assessed as shown in Fig. 4. Polar to equatorial ratio (P:E) ranges represent the minimum and maximum values found among species in each genus. The Myosotis outgroup species are included as well. Page 16.

Table 2. Cryptanthinae taxa sampled with Herbarium and accession information. Abbreviations are as follows: NCC - North Coast California, UC/JEPS – University of California at Berkeley/Jepson, UAZ – University of Arizona, CONC – Universidad de Concepción Peru, MO – Missouri Botanical Gardens. Page 51-53.

Table 3. Pollen shape and subshape distribution among 74 taxa in the Cryptanthinae. The taxa are organized with regard to Erdtman (1966) shape as well as Faegri and Iversen (1975) subshape. The three different shapes are based on P:E ratios. The subshapes are based on the measurements taken in Fig. 4. The Myosotis outgroup species are included as well. Page 80.

Table 4. Pollen aperture types found across 74 taxa of the Cryptanthinae. The Myosotis outgroup species are included as well. Page 81.

Table 5. Presence or absence of transverse grooves in 74 taxa of Cryptanthinae pollen. The Myosotis outgroup species are included as well. Page 81.

Table 6. Presence or absence of polar pseudo-apertures across 74 taxa of Cryptanthinae pollen. The Myosotis outgroup species are included as well. Page 82.

Table 7. Sculpturing types for pollen of 74 taxa in the Cryptanthinae. The Myosotis outgroup species are included as well. Page 82.

Table 8. Habitat moisture level for 74 Cryptanthinae taxa and two Myosotis outgroup taxa. Page 83.

Table 9. Flowering period in selected Cryptanthinae taxa. Page 83.

Table 10. Table 10. Average polar and equatorial diameter, P:E ratio, standard deviation of P and E. A standard test of normality for both P and E was performed in excel and all were normal with a significance of p<.05. Page 84-85.

viii

List of Figures

Figure 1. General flowering characteristics of members of the Boraginoideae including cymose-based terminal inflorescence, radial corolla, superior ovary, epipetalous stamens, and nutlets (Watson and Dallwitz 2013). Page 2.

Figure 2. Current placement of the Boraginaceae in the Boraginales clade (APGIII 2014). Page 3.

Figure 3. Recent phylogeny of the Cryptanthinae. ‘Maximum likelihood (ML) analysis with bootstrap values (≥ 70%) indicated at nodes’ (Hasenstab-Lehman and Simpson 2012). Note the polyphyletic dispersion of Plagiobothrys and Cryptantha. Page 5.

Figure 4. Pollen measurements to determine sub-shape: polar length (P), equatorial width (E), polar width (A), distance from outer polar width to maximum width (B), length from maximum width to pole (C), distance between outside maximum width (D), and maximum width (W). These line drawings show the most commonly observed subshapes, but are not representative of all the variation present. Page 11.

Figure 5. Faegri and Iversen (1975) sub-shape classes with grayed-in sub-shapes representing those found in the Cryptanthinae subtribe. Page 12.

Figure 6. Molecular phylogeny for the Cryptanthinae subtribe (Guilliams 2013) used throughout this study with era, period, and epoch information added to the figure (Polly et al. 2011). The scale axis at the bottom of the figure is in millions of years. Genera abbreviations: Pl. – Plagiobothrys; Pe. – Pectocarya; H. – Harpagonella; C. – Cryptantha; A. – Amsinckia; M. – Myosotis are used throughout this report. Page 13.

Figure 7. Pollen size variability across genera in the Cryptanthinae ranges from 40.85µm-4.85µm in polar length. Maximum and minimum sizes are represented for each genus. Species are as follows: A. A. vernicosa, B. A. menziesii, C. M. discolor, D. Pl. albiflorus, E. Pe. pusilla, F. C. confertiflora, G. M. laxa, H. Pe. setosa, I. C. muricata, J. Pl. humilis. See Table 1 for sizes. Page 17.

Figure 8. The range of Erdtman (1966) pollen shapes in the Cryptanthinae for Plagiobothrys, Pectocarya, and Cryptantha based on P:E ratio. Scale bars for each image are included. Species are as follows: A. Pl. uncinatus, B Pl. tenellus, C. Pe. linearis var. feroculata, D. Pl. kingii var. kingii, E. C. costata, F. Pl. mollis var. mollis, G. Pl. hispidulus. Page 18.

Figure 9. Pollen subshapes in the Cryptanthinae. Species are as follows: A. A. menziesii, B. Pe. setosa, C. Pl. infectivus, D. Pl. procumbens, E. Pl. uncinatus, F. Pl. verrucosus, G. Pl. collinus var. collinus, H. C. circumscissa, I. Pe. anomala, J. A. furcata, K. Pl. humilis, L. Pe. brachycera. * denotes asymmetrical pollen. Page 19.

Figure 10. Three different aperture types in Cryptanthinae pollen. The species above are: A. Pl. scouleri, B. A. retrorsa, C. C. flavoculata. The image also shows the difference between pollen without a continuous transverse groove (A and B) and pollen that has a transverse groove (C). Page 20.

Figure 11. Presence or absence of polar pseudo-apertures in Cryptanthinae pollen. Species above are: A. C. micromeres and B. Pl. figuratus. Page 21.

Figure 12. Pollen in the Cryptanthinae has 5 different types of sculpturing. The taxa and sculpturing types above are as follows: A. Pe. platycarpa, B. C. flaccida, C. H. palmeri var. arizonica, D. C. gracilis, E. A. spectabilis. Page 22.

Figure 13. The Cryptanthinae phylogeny (Guilliams 2013) split into major clades (I and II) and subclades (IIA, IIB) for discussion were visualized in Mesquite (Maddison and Maddison 2011). Pie charts at nodes indicate maximum likelihood of the ancestral state. Page 23.

Figure 14. Pollen shape (Erdtman 1966) painted on the Cryptanthinae tree (Guilliams 2013). Character states are shown in upper left. The basal node (indicated by arrow) described in text. Page 25.

Figure 15. Pollen subshapes (Faegri and Iversen 1975) painted on the Cryptanthinae phylogeny (Guilliams 2012). Character states are shown in upper left. Key nodes (arrows 1-3) described in text. Page 27.

Figure 16. Pollen aperture types on Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-3) described in text. Page 29.

Figure 17. Pollen sculpturing mapped on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-4) described in text. Page 31.

Figure 18. Habitat moisture displayed on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1 and 2) described in text. Page 33.

Figure 19. Range distribution on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-5) described in text. Page 35.

Figure 20. Mirror tree (Maddison and Maddison, 2011) of Cryptanthinae with pollen subshape (left) and habitat moisture (right). Subshape abbreviations follow Fig. 15. Page 37.

Figure 21. A selection of Scanning Electron Microscope images of Cryptanthinae pollen taken for this study. The images below have not been altered for size or contrast. Pollen shown in each image may not necessarily be the Archetype, but instead are part of the natural size variation within each taxon. Images are listed alphabetically. Scale bars vary. Legend for SEM images is shown below. Page 54-66.

Figure 22. Line drawings (equatorial view left, polar view right) generated for Cryptanthinae pollen archetypes based on the measurements shown in Fig. 4. Name of taxon, P:E ratio, and subshape are listed for each drawing. Abbreviations for genera are: Pl. – Plagiobothrys; Pe. – Pectocarya; H. – Harpagonella; C. – Cryptantha; A. – Amsinckia; M. – Myosotis. Abbreviations for subshapes are: co - compressed oval, coc - constricted oval circular, cr - compressed rectangular, o - oval, do - depressed oval, b - biconvex, rh - rhomboidal. Key for figures is listed below. Page 67-79.

Figure 23. The original Guilliams (2013) molecular phylogeny with bootstrap values at nodes. Scale axis at the bottom of the figure is in millions of years calibrated by ITS mutation rates (Kay et al. 2006). Page 86.

Introduction Many scientists strive to solve the mysteries surrounding evolutionary history using phylogenetic reconstruction. Historically, these ‘trees of life’ were devised using the morphological characteristics of organisms. Within the last three decades, scientists have started to use molecular characteristics to tease out evolutionary history in order to minimize erroneous, homologous relationships. Molecular data are more highly conserved than phenotypic data, and provide a more accurate representation of relatedness (Singh 2004). Using molecular data has forced the reassessment of many lineages, and allowed scientists to clarify some groups into more parsimonious clades.

For example, morphologically-based mammalian phylogenies disagree with respect to the location of many taxa depending on which morphological features were used to generate the phylogeny. However, when combined with a molecular phylogeny, the morphological characteristic of placentation type seems to generate additional support for taxa branch placement (Lee and Camens 2009). This could revolutionize the way taxonomists treat morphological characters. Physical traits become useful for uncovering evolutionary selection pressures that lead to diversity.

Members of the Boraginaceae are herbaceous annuals or perennials (Zomlefer

1994). Currently this family is listed as having 110 genera and 1,595 species (APGIII

2014). Their inflorescences are cymose and terminal (APGIII 2014). Flowers consist of a five-lobed, sympetalous (fused) corolla, five distinct or basally connate (fused) sepals, five epipetalous stamens (attached to petals), and a two-carpellate superior ovary with false septa, making them four-locular (chambered) (Zomlefer 1994). Fruits contain one to four nutlets (Baldwin et al. 2012), and are modernly referred to as a loculicidal capsule

(a dry-dehiscent fruit) (APGIII 2014) (Fig. 1). Recent work on the phylogeny of the

Boraginaceae and related families suggests the family should be expanded to include other recognized plant families, such as the Hydrophyllaceae. The expanded view of the

Boraginaceae places taxa described by Zomlefer (1994) in the subfamily Boraginoideae.

The Boraginoideae subfamily’s main characteristics are circinate, scorpioid cymes (Buys and Hilger 2003), gynobasic styles, a deeply four-lobed ovary, and a fruit of four distinct schizocarp nutlets (Al-Shehbaz 1991; Gottschling et al. 2001).

The Boraginaceae, along with six other families, comprise the Boraginales. The

Boraginales are members of the Asterid 1 clade which is notable in that it contains many subdivisions that have been identified as non-monophyletic (Fig. 2) (APGIII 2014).

Figure 2. Current placement of the Boraginaceae in the Boraginales clade (APGIII 2014).

Problems with phylogenetic resolution are found within the Boraginaceae. The

Boraginoideae, a subfamily within the Boraginaceae, also appears to have problematic phylogenetic regions. Scientists have been taking particular interest in this subfamily as of late. Using molecular evidence, studies have found deep clades in the Boraginoideae that correspond to the tribes Echiochilieae, Lithospermeae, Cynoglosseae, Borgineae, and a proposed tribe Codoneae (Langstrom and Chase 2002; Nazaire and Hufford 2013).

Formerly recognized Boraginoideae tribes included Eritrichieae (Bentham and Hooker

1873; Gürke et al. 1897), Myosotideae, and Trigonotideae (Takhtajan 1997). These classical tribes were based on morphological characteristics. Recent molecular studies have shown these three tribes are actually nested within the Cynoglosseae (Langstrom and Chase 2002; Weigend et al. 2010; Mozaffar et al 2013).

Phylogenetic relationships in the Boraginoideae also require resolution at the genus level. Three of these genera (Amsinckia, Cryptantha, and Plagiobothrys) have long been categorized in the Eritricheae based on morphological characteristics (Bentham

4 and Hooker 1873; Gürke et al. 1897). Currently, they have been placed in the subtribe

Cryptanthinae within the Cynoglosseae based on molecular data (Langstrom and Chase

2002; Weigend et al. 2010; Hasenstab-Lehman and Simpson 2012; Mozaffer et al. 2013).

A molecular reconstruction of a large portion of the Cryptanthinae subtribe focusing on

Cryptantha recently found the genus to be polyphyletic (Fig. 3; Hasenstab-Lehman and

Simpson 2012). This study also found Plagiobothrys to be polyphyletic, with species now spread across the genera Allocarya, Amsinckiopsis, and Plagiobothrys (Hasenstab-

Lehman and Simpson 2012). Further analysis of the Plagiobothrys was suggested.

Within this particularly convoluted subtribe, pollen variation has been found to have great utility for systematics (Hargrove and Simpson 2003). Pollen type in the

Cryptanthinae varies among genera (Scheel et al. 1996; Cohen 2011). Species and subspecies level taxonomy in this subtribe are also eurypalynous (Diez and Valdez 1991).

Eurypalynous taxa exhibit variability in size, shape, texture, and aperture types (Naik

1984).

Pollen morphology has been used to discern taxonomic relationships among fossilized plants where other forms of plant material are not well preserved. When combined with molecular data, these taxonomic relationships become more strongly supported (Xu et al. 2011). There are limitations to the usefulness of pollen analysis for the application of phylogenetic reconstruction. This is due primarily to degradation of material as well as the amount of time required for accurate identification of taxa.

Typically pollen has been used to make generalizations regarding geographic climate reconstruction, but specimens are identified to the family or genus level only (Ortega-

Rosas et al. 2008; Lazarova et al. 2012). More in-depth studies have had success developing taxonomic keys to identify plants to the species level using pollen morphology (Scheel et al. 1996; Falatoury et al. 2011; Hasenstab-Lehman and Simpson

2012).

Pollen of monocots is typically monoporate, whereas the pollen of all eudicots has three openings. This is due to strong selection pressure for more apertures – a greater number of apertures increases reproductive success by providing multiple routes for germination (Furness and Rudall 2004). Other traits selected for include dispersion, drought tolerance, pollination, and fertilization (Falatoury et al. 2011). There are two different types of openings pollen can have: pores or slits (colpi). Various combinations of these aperture types in Boraginaceae pollen have been observed by prior research.

These combinations include tricolporate (having three colpi, each with pores) (Erdtman

1966), heterocolpate (having 6 colpi, 3 of which also have pores) (Kapp 1969), or

7 stephanocolporate (synonym for zonocolporate - pollen have 6 colpi, all of which have pores) (Kapp 1969). In each of the cases where 6 apertures are present, the pollen has three true apertures and three pseudo-apertures. The three true apertures can be identified by exine deposits (Erdtman 1966). Pseudo-apertures resemble apertures, but presumably do not function as an exit site for a pollen tube. Instead, they provide areas where folding occurs to prevent water loss without damaging structural integrity (Volkova et al. 2013).

Studies examining the morphology of pollen within the Boraginaceae have yielded valuable information for the appropriate identification of many taxa in this family

(Hilger et al. 2004; Biznet et al. 2010; Falatoury et al. 2011, Fukuda and Ikeda 2012).

Scheel et al. (1996) found that of 30 South American taxa, species level distinctions could be categorized into nine pollen types, and suggested re-evaluation of the systematics of this group.

One of the purposes of this study was to determine the range of variation in pollen structure in the subtribe Cryptanthinae by sampling taxa from the genera Amsinckia,

Cryptantha, Harpagonella, Pectocarya, and Plagiobothrys. Myosotis was chosen as the outgroup, a member of the Lithospermeae tribe (Bentham and Hooker 1873; Gürke et al.

1897; Takhtajan 1997). Previous study has indicated the Cryptanthinae is a sister group to Myosotis, making it an appropriate outgroup candidate (Fig. 3; Hasenstab-Lehman and

Simpson 2012).

Using molecular data has forced the reassessment of many lineages, and allowed scientists to clarify some groups into more parsimonious clades. For example, long standing morphologically-based placental mammalian phylogenies appear riddled with homoplasy when compared to their molecular counterpart. However, when the two trees

8 are combined using a total evidence approach, morphological characters generate additional support for the molecularly based phylogeny (Eernisse and Kluge 1993; Lee and Camens 2009). Huang et al. (2013) found that combining molecular and morphological evidence for the genus Lappula (Boraginaceae) generated a better resolved phylogeny than one based exclusively on genetic evidence. This type of analysis was also successful for Lithospermum L. (Boraginaceae) (Cohen 2011). An in depth analysis of eight Cryptantha taxa within the subfamily Boraginoideae yielded not only valuable taxonomic information, but also inferred how the evolution of pollen ultrastructure and aperture formation are correlated (Hargrove and Simpson 2003).

Another goal of this study was to analyze whether pollen morphological characteristics in the Cryptanthinae add support to a molecular phylogeny. Studies in the past have been successful at overlaying morphological characters on molecular phylogenies (Eernisse and Kluge 1993; Hargrove and Simpson 2003; Lee and Camens

2009; Biznet et al. 2010; Cohen 2011; Fukuda and Ikeda 2012; Hasenstab-Lehman and

Simpson 2012; Huang et al 2013). Molecular data are more highly conserved than phenotypic data, and provide a more accurate representation of relatedness (Singh 2004).

In addition to providing phylogenetic support, it may be that pollen morphology in conjunction with molecular data can elucidate evolutionary selections pressures from ecological or biogeographic features for members in the Cryptanthinae. The total evidence approach was successful for understanding evolutionary trends in ecological diversity for Selaginella (Arrigo et al. 2103). Lu et al. (2013) found that by combining morphological and molecular data, they could infer global dispersal patterns in the

Vitaceae. This type of analysis is especially helpful in cases where a trait has arisen

9 several times, or where taxa have an amphitropical distribution. A strictly morphological tree would put homologous species together in clades that conflict with molecular data.

Distributing physical attributes of Lebeoninae (Cyprinidae), a subfamily of cyprinid fishes, on a molecular tree showed homplasticity in the group, and suggested dispersal patterns for this group (Zheng et al. 2012). Going one step further, this type of analysis was used to infer ecological limits, namely temperature and natural barriers such as water, that restrict range expansion in the Solanaceae, Verbenaceae, and Bignoniaceae

(Olmstead 2013). Grimm and Almeda (2013) went so far as to infer natural disasters that may have contributed to speciation events based on dating molecular divergence in

Chaetolepis (Melastomataceae).

Research Questions 1. What is the range of variation in pollen structure in subtribe Cryptanthinae

(Boraginaceae)?

2. Does overlaying pollen morphological characteristics increase support for the

molecular-based phylogeny with a subset of Cryptanthinae taxa?

3. Does pollen morphology show a correlation with ecological or biogeographic

features of members of Cryptanthinae?

Methods Pollen of 74 species from five genera (Amsinckia, Harpagonella, Cryptantha,

Plagiobothrys, Pectocarya) and two outgroup species of Myosotis were surveyed

(Appendix 1). Pollen was collected from anthers of herbarium specimens. Specimens came from the North Coast Herbarium of California (NCC), University of California

Berkeley/Jepson Herbarium (UC/JEPS), Universidad de Concepción Herbarium

(CONC), the Missouri Botanical Garden Herbarium (MO), and the University of Arizona

Herbarium (UAZ). Using microscopically sharpened tweezers and a dissecting microscope, anthers were taken from four separate flowers on each herbarium sheet.

This was to ensure mature grains were obtained, as younger or older pollen have a higher occurrence of pollen deformation. When available, flowers were selected from multiple plants on an accession.

Samples were mounted on 260 µm Carbon Conductive Pelco Image Tabs™ (Ted

Pella Inc., Redding, CA) which are ideal for small particles such as pollen and insect parts. Samples were then sputter coated with a 2-5 angstrom thick Au/Ni mix (Pelco

Sputter Coater 91000 Model 3). Images were taken on a minimum of 20 pollen grains using a Hitachi S-3000N Scanning Electron Microscope in the William Keck

Microanalysis Laboratory at Sonoma State University. Pollen in the appropriate orientation was measured using Quartz PCI and Adobe Photoshop CS5. Images that were not used for size measurements because of orientation were used to examine various other morphological features, such as the presence or absence of polar pseudo-apertures.

Data were collected for polar (P) and equatorial (E) distances, aperture type

(heterocolpate, zonocolpate, or zonoporate), aperture number, sculpturing (fossulate, foveolate, echinate, gemmate-clavate, or reticulate), presence or absence of a transverse groove, and the presence or absence of polar pseudo-apertures. Pollen terminology is consistent with Punt et al. (2007).

Size distribution of P and E distances were tested for normality in Microsoft

Excel in each taxon. The average size for each taxon was used to generate an archetype, or ideal pollen size to compare with the other taxa. The P:E ratio was based on the archetype pollen and used as a proxy for shape (Erdtman 1966). The three shapes observed from this approach were subprolate, prolate, and perprolate. However, within each of these three shape categories, variation was also observed so a second classification for sub-shape was used in conjunction with the Erdtman shapes. Sub-shape was classified by generating line drawings from measurements of the archetype pollen in each taxon (Fig. 4). These line drawings were compared to a set of shapes generated by

Faegri and Iversen (1975) because their range of variability covered the diversity observed in the Cryptanthinae subset (Fig. 5).

Figure 5. Faegri and Iversen (1975) sub-shape classes with grayed-in sub-shapes representing those found in the Cryptanthinae subtribe.

A phylogenetic tree of the subtribe Cryptanthinae (Boraginoideae), based on molecular data, was provided for this study by M. Guilliams (Guilliams 2013) from an on-going investigation into the phylogeny and evolution of the Boraginoideae. The tree

(Fig. 6) was compiled using approximately 1,400 base pairs from the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of the nuclear ribosomal DNA repeat. A Bayesian analysis was performed in BEAST (Drummond and Rambaut 2007), with Myosotis taxa as the outgroup. Tree maximum clade credibility was set up using

TreeAnnotater in BEAST, and visualized using FigTree (Rambaut 2006), with posterior probabilities mapped on the branches (data not shown). Time was calibrated by enforcing rates of evolution based on ITS (Kay et al. 2006). The Guilliams tree is used as the template for all trees in this study.

Pollen characteristics were mapped on the molecular phylogeny using Mesquite

(Maddison and Maddison 2011) to observe changes in discrete character states. Since branch lengths were known, they were incorporated to perform a Maximum Likelihood

(ML) analysis (Schluter et al. 1997) to determine ancestral character states. Trees were visualized using the ball and stick method, with pie charts at each node indicating the proportional likelihood of ancestral characteristics.

Pollen P length was used as a proxy for size to observe broad trends in pollen size on the phylogeny. Continuous pollen length measurements were converted into size classes for input into Mesquite. The size categories used for this visualization were

<6.00µm, 6.01µm-8.00µm, 8.01µm-10.00µm, 10.01µm-20.00µm, and >20.01µm. These ranges were chosen based on observed breaks in the continuous data set. All other characteristics mapped on the phylogeny were discrete. Sculpturing classification follows Glalrini and Ricciardelli D’Albore (1998).

Biogeographic distribution (North America, South America, or both), habitat moisture (wet, dry), and breeding system (homostylous, heterostylous) were obtained from Horn (2000) and Baldwin et al. (2012). Flowering period (spring, summer) data were collected from the CCH (2010). If taxa were not listed in the Consortium database, data were acquired from accession labels of the specimens sampled. The flowering period data were then placed into Spring or Summer to produce a binary character for analysis. In the Northern Hemisphere, plants flowering in March, April, or May were placed into the category Spring, and plants flowering in June, July, and August were combined into Summer. In the Southern Hemisphere, plants flowering in September,

October, and November were joined into Spring, and December, January, and February

15 flowering plants were combined into Summer. The final trait examined was a type of breeding system known as hercogamy. Hercogamous plants have a structural separation of male and female flower reproductive parts that enhances outbreeding (Simpson 2006).

Though several types of hercogamy exist, the specific type looked at in this study is heterostyly, or the discrepancy in relative lengths of anthers and stigma (Darwin1877).

Homostylous plants have pistils and anthers with the same length. Heterostylous flowers are sexually dimorphic with anthers and pistils differing in length, which facilitates reciprocal fertilization (Darwin 1877).

Comparisons were performed between morphological and biogeographic traits to determine underlying evolutionary selection pressures responsible for high pollen morphological diversity. This was done by generating mirror trees in Mesquite

(Maddison and Maddison 2011), and looking patterns amongst all scored characteristics.

Results

Morphological Variation Observed in the Cryptanthinae (Boraginaceae) Scanning Electron Microscope images (Appendix 2) in conjunction with archetype line drawings (Appendix 3) of pollen for each taxon shows that high morphological diversity is prevalent in the Cryptanthinae. Variability was observed in all of the following categories: size, shape, subshape, apertures, and sculpturing.

Pollen Size Within the observed taxa in the subtribe Cryptanthinae, pollen exhibits a large range of size variation (Table 1, Fig. 7). They have a polar length ranging from 4.849µm-

40.85µm, and an equatorial width ranging from 1.681µm-25.60µm. Amsinckia has the

16 largest pollen in the subtribe, at an order of magnitude over all the other Cryptanthinae genera (Fig. 7A, B). Of the remaining genera, two Harpagonella taxa show the least variation in polar length with a difference of 0.753µm and were therefore not shown in

Fig. 7. The greatest variability in polar length is found in Plagiobothrys with a difference of 6.301µm (Fig. 7D, J). The two taxa in the outgroup Myosotis also have high size variability in polar length with a difference of 8.124µm (Fig. 7C, G). Cryptantha and

Pectocarya have a moderate amount of polar length variability with a difference of

3.82µm and 2.89µm respectively (Fig. 7E, F, H, I). Of the Cryptanthinae genera,

Cryptantha has the greatest variability in equatorial width with a difference of 4.526µm

(Fig. 7F, I). Harpagonella is the least variable in equatorial diameter with a difference of

1.906µm (not shown). Plagiobothrys and Pectocarya have a moderate amount of variability with a difference of 3.30µm and 3.47µm respectively (Fig. 7D, E, H, J).

Genus Number of taxa P (µm) E (µm) P:E examined Min-Max Min-Max Min-Max

Amsinckia 7 22.18-40.85 17.40-25.60 1.27-1.70

Cryptantha 11 5.543-9.368 1.921-6.447 1.40-2.90

Harpagonella 2 7.107-7.860 3.290-5.196 1.50-2.16

Myosotis 2 8.836-16.96 4.986-11.28 1.50-1.80

Pectocarya 8 6.476-9.639 3.290-6.765 1.30-2.16

Plagiobothrys 46 4.849-11.15 1.681-4.989 1.19-3.31 Table 1. Pollen size variation observed across genera in the subtribe Cryptanthinae (Boraginaceae). Polar (P) and equatorial (E) lengths were assessed as shown in Fig. 4. Polar to equatorial ratio (P:E) ranges represent the minimum and maximum values found among species in each genus. The Myosotis outgroup species are included as well.

µm

The outgroup Myosotis has a high amount of variability in equatorial distance with a difference of 6.294µm indicating the variability exists at higher taxonomic levels as well

(Fig. 7C, G).

Pollen Shape

P:E ratios were used to determine pollen shape following Erdtman (1966). This classical method yielded three shape categories; subprolate (1.14-1.33), prolate (1.33-

2.0), and perprolate(>2.0) (Appendix 4). P:E ratios in Cryptanthinae pollen vary from

1.19-3.31, and are most variable among Plagiobothrys which exhibits both of those extremes (Table 1, Fig. 8). Plagiobothrys and Pectocarya pollen exhibit three shapes; subprolate, prolate, and perprolate. Amsinckia pollen is either subprolate or prolate.

Cryptantha and Harpagonella pollen is either prolate or perprolate. The pollen of the

Myosotis outgroup is prolate for the two species examined.

Pollen Subshape

Pollen within the Cryptanthinae displays seven of the eleven Faegri and Iversen

(1975) sub-shapes, including one asymmetrical form (Appendix 3, 4; Figs. 5, 9).

Amsinckia taxa are rhomboidal, oval, or depressed oval (Fig. 9A, J). Cryptantha taxa are constricted oval circular, depressed oval, compressed oval (Fig. 9H). Plagiobothrys taxa are constricted oval circular, compressed oval, constricted rectangular, and depressed oval (Fig. 9C-G, K). Harpagonella taxa are compressed oval and depressed oval (not shown). Pectocarya species exhibited the highest amount of subshape variation, having compressed oval, constricted oval circular, biconvex, oval, and compressed oval forms present (Fig. 9B, I, L). Pectocarya brachycera and Pectocarya anomala both have asymmetrical pollen (Fig. 9I, L).

Pollen Apertures

Cryptanthinae pollen had three distinct aperture types; heterocolpate, zonocolpate, and zonoporate (Appendix 5, Fig. 10). Heterocolpate is the most frequently observed type. Plagiobothrys taxa exhibit all three types. Cryptantha and Pectocarya pollen have either heterocolpate or zonoporate apertures. Amsinckia and Harpagonella pollen have either heterocolpate or zonocolpate apertures. The pollen of Myosotis outgroup taxa have only heterocolpate apertures.

Another morphological distinction includes the presence or absence of a transverse groove in Cryptanthinae pollen (Appendix 6, Fig. 10). They are present in two taxa of Cryptantha, two taxa of Pectocarya, and three taxa of Plagiobothrys. Both

Harpagonella taxa have transverse grooves. Transverse grooves do not occur in the

Amsinckia taxa surveyed or the Myosotis outgroup taxa.

In addition to equatorial pseudo-apertures, some Cryptanthinae pollen also possesses polar pseudo-apertures (Appendix 7; Fig. 11). Pollen of almost half the taxa in

Cryptantha, Plagiobothrys, and Pectocarya has polar pseudo-apertures (Fig. 11B), whereas the other half do not (Fig. 11A). Taxa in Harpagonella and Amsinckia lack this feature, but taxa in the Myosotis outgroup exhibit variability in this trait.

Figure 11. Presence or absence of polar pseudo-apertures in Cryptanthinae pollen. Species above are: A. C. micromeres and B. Pl. figuratus.

Pollen Sculpturing

Cryptanthinae pollen sculpturing has various textures including fossulate, foveolate, echinate, gemmate-clavate, and reticulate forms (Appendix 8, Fig. 12).

Echinate is the least observed texture, found only in Pectocarya platycarpa (Fig. 12A).

All Amsinckia taxa are reticulate (Fig. 12E). Two species of Cryptantha are gemmate- clavate (Fig. 12D), one is foveolate (Fig. 12C), and the remaining eight are fossulate textured (Fig. 12B). Plagiobothrys, Harpagonella, and the Myosotis outgroup had either fossulate or foveolate surfaces (Fig. 12B, C).

Phylogenetic Trait Distribution

The Cryptanthinae tree was divided into two major clades to facilitate presentation of results (Fig. 13). Clade I is made up of 43 Plagiobothrys taxa. Clade II is made up of two subclades. The first (Clade II-A) is made up of Pectocarya,

Harpagonella, and Cryptantha taxa. The two Harpagonella taxa are nested within

Pectocarya taxa. Clade II-B is comprised of Amsinckia and three Plagiobothrys taxa.

Myosotis taxa are the Outgroup.

Pl. parishii Pl. bracteatus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gracilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infectivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 13. The Cryptanthinae phylogeny (Guilliams 2013) split into major clades (I and II) and subclades (IIA, IIB) for discussion were visualized in Mesquite (Maddison and Maddison 2011). Pie charts at nodes indicate maximum likelihood of the ancestral state.

Pollen Size

Large pollen is found exclusively in Amsinckia (Table 1, Fig. 7A, B). Pollen size data for all other taxa examined do not increase phylogenetic resolution in the

Cryptanthinae subset.

Pollen Shape

Clade I is primarily comprised of perprolate pollen (Fig. 14). Clade II is dominated by prolate pollen. The outgroup also consists of prolate pollen. Subprolate appears as a derived condition restricted to two taxa in each clade. In Clade I, the two subprolate taxa are in a sister group, but in Clade II this condition is derived in distantly related taxa, once in Clade II-A, and once in Clade II-B. The basal node (see arrow on

Fig. 14) shows a nearly equal likelihood for each of the Erdtman (1966) shapes present in the Cryptanthinae to be the ancestral state (subprolate – 32.86%, prolate – 35.89%, perprolate – 31.25%).

Pl. parishii Pl. bracteatus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. gree ne i Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infec tivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. microme res C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 14. Pollen shape (Erdtman 1966) painted on the Cryptanthinae tree (Guilliams 2013). Character states are shown in upper left. The basal node (indicated by arrow) described in text.

Pollen Subshapes

At the basal node, each Faegri and Iversen (1975) subshape has a roughly 14% chance of being the ancestral state (see arrow 1 on Fig. 15). Clade I arose around 15-20 million years ago during the Miocene (Fig. 6). One of the first groups to branch off in

Clade I is the monophyletic group consisting of Plagiobothrys uncinatus, Pl. verrucosus, and Pl. tenellus which all have depressed oval pollen. The node at the base of this group shows a 66% ML that the ancestor was depressed oval (see arrow 2 on Fig. 15).

However, most of the remaining taxa in Clade I shows little variability in subshape, being dominated by the constricted oval circular(coc) form (Fig. 15). The node indicated by arrow 3 on Fig.15 shows a 92% ML that the ancestor was coc. After this point, subshape diversity drastically decreases. Interestingly, the timing of divergence for this group coincides with the transition period between the Miocene and the Pleistocene (Fig. 6).

Clade II arose much earlier, towards the end of the Oligocene (Fig. 6). Clade II exhibits a high amount of diversity in subshape form, with members of six different categories present.

Pl. parishii Pl. brac te atus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifoli us Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infectivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumsciss a A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 15. Pollen subshapes (Faegri and Iversen 1975) painted on the Cryptanthinae phylogeny (Guilliams 2012). Character states are shown in upper left. Key nodes (arrows 1-3) described in text.

Pollen Apertures

Pollen aperture type in the Cryptanthinae is primarily heterocolpate (Fig. 16), with a likelihood of 92.56% being the ancestral state for the subtribe (see arrow 1 on Fig.

16). However, pollen in a few clades derived zonoporate or zonocolpate pollen apertures.

Zonoporate pollen appears novelly derived in one taxon in Clade I, and three taxa in

Clade II-A. Two of the taxa in Clade II-A form a sister group: Cryptantha flavoculata and C. confertiflora (see arrow 2 on Fig. 16). The node at arrow 2 indicates the ancestor these taxa arose from was probably zonoporate as well (ML zonoporate - 66%). The other taxon in Clade II-A with zonoporate pollen is Pectocarya penicillata.

Zonocolpate arose novelly in two unrelated taxa in Clade I and one taxon in Clade

II-A (Fig. 16). The two major clades of Amsinckia share a genetic link that dates back to the Miocene (Fig. 6). However, extant members of both major clades in this group arose during the Pleistocene (Fig. 6). Heterocolpate Amsinckia taxa arose earlier than the rest of Clade II-B, approximately 5-10 million years ago (Fig. 6). Zonocolpate Amsinckia was derived in this clade less than 5 million years ago (Fig. 6, arrow 3 on Fig. 16). The

ML value at this node shows a 99% chance that the ancestor for this group was zonocolpate.

Transverse grooves and polar pseudo-apertures were uninformative in the context of the Cryptanthinae phylogeny (trees not shown). Both traits permeate throughout the phylogeny and were derived multiple times. Polar pseudo-apertures are the more labile of the two characteristics.

Pl. parishii Pl. bracteatus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infectivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. re tror sa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 16. Pollen aperture types on Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-3) described in text.

Pollen Sculpturing

Pollen with little (foveolate) or no (fossulate) sculpturing predominates members of the subtribe as well as the outgroup (Fig. 17). The combined likelihood of little to no sculpturing at the base of the subtribe is 53.60%, indicating this is probably the ancestral condition of the subtribe (see arrow 1 on Fig. 17). Each of the other sculpturing forms has a likelihood ratio of less than 16% at the basal node of the subtribe (see arrow 1 on

Fig. 17). Clade I is exclusively one condition or the other. The majority of variability in sculpturing is shown in Clade II. All Amsinckia taxa have a reticulate surface, which evolved during the Pleistocene (Fig. 6). The ML value where this group diverged indicates a 99% chance that the ancestor of all Amsinckia also had reticulate sculpturing

(see arrow 2 on Fig. 17).

Other forms of sculpturing exist in Clade II-B such as a group of two related, but not monophyletic Cryptantha taxa having gemmate-clavate pollen. These two taxa, C. gracilis and C. micromeres have persisted since the Miocene (Fig. 6). The node at the base of this clade (shown by arrow 3 on Fig 17) indicates 39% likelihood that the common ancestor had fossulate sculpturing, and 36% likelihood that it had gemmate- clavate sculpturing. The final form of sculpturing observed in this group was only found in Pectocarya platycarpa, which had an echinate surface. This taxon evolved shortly after the end of the Pliocene (Fig. 6). The ancestor that unifies this clade (see arrow 4 on

Fig 17) has 51% chance of being fossulate and 38% chance of being echinate.

Pl. parishii Pl. bracteatus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflor us Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acil is Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infectivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 17. Pollen sculpturing mapped on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-4) described in text.

Habitat, Biogeography, and Breeding System

Habitat Moisture

Cryptanthinae habitat moisture shows a distinct split on the phylogeny (Fig. 18).

Dry habitats appear to be ancestral in the subtribe as indicated by the basal node where

ML dry is 95.68% and wet is 4.32% (see arrow 1 on Fig. 18). All members of Clade II and the basally derived members of Clade I are found in dry habitats. Occurrence in moist habitats is a derived condition in Clade I, which appears during the transition period between the Miocene and the Pleistocene (Fig. 6, see arrow 2 on Fig. 18).

Pl. parishii Pl. bracteatus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. s tr ictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infec tivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linearis Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 18. Habitat moisture displayed on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1 and 2) described in text.

Range

The subtribe occurs primarily in North America, which appears to be the ancestral condition (ML North – 52.90%, South – 22.53%, Both – 24.57%) (see arrow 1 on Fig.

19). Within Clade I, there appears to be two instances of dispersal to South America. In both cases, a single and apparently derived taxon has dispersed back to North America

(see arrows 2-5 on Fig. 19). Clade II appears more variable in the character distribution.

Several range extensions have occurred to include both North and South America, and range dispersion appears to have happened in both the forward and reverse directions.

Pl. parishii Pl. brac te atus Pl. glyptocarpus var modes tus Pl. hispidulus Pl. tener var tener Pl. cusickii Pl. diffusus Pl. scouleri Pl. cognatus Pl. corymbosus Pl. calandrinoides Pl. albiflorus Pl. scriptus Pl. undulatus Pl. figuratus Pl. humilis Pl. congestus Pl. linifolius Pl. kunthii Pl. m ollis Pl. procumbens Pl. collinus v collinus Pl. uliginosus Pl. humistratus Pl. acanthocarpus Pl. distantiflorus Pl. glyptocarpus var. glyptocarpus Pl. austinae Pl. greenei Pl. strictus Pl. canescens va r catalinensis Pl. canescens va r c ane sce ns Pl. arizonicus Pl. uncinatus Pl. verrucosus Pl. tenellus Pl. collinus va r fulve scens Pl. pringlei Pl. collinus va r gr acilis Pl. collinus va r californicus Pl. collinus va r urs inus Pl. infectivus Pl. fulv us Pe. penicillata Pe. dimorpha Pe. platycarpa Pe. linear is Pe. anomala Pe. brachycera H. palmeri va r palm eri H. palmeri va r ar izonic a Pe. se tos a Pe. pusilla C. flavoculata C. confertiflora C. simulans C. flaccida C. gracilis C. micromeres C. muricata C. maritima C. costata C. racemosa C. circumscissa A. spectabilis A. intermedia A. menziesii A. eastwodiae A. retrorsa A. furc ata A. vernicosa Pl. kingii var har ke ns ii Pl. kingii var kingii Pl. jonesii M. discolor M. lax a Figure 19. Range distribution on the Cryptanthinae phylogeny (Guilliams 2013). Character states are shown in upper left. Key nodes (arrows 1-5) described in text.

Flowering Period

Flowering period is also labile between Spring and Summer across the phylogeny, with a higher concentration of Summer flowering plants occurring at the most recently derived portion of Clade I (Appendix 9). Amsinckia, however, are strictly Spring flowering.

Breeding System

A few Cryptanthinae taxa exhibit the atypical feature of heterostyly (tree not shown). The taxa exhibiting this trait are Cryptantha flavoculata, Cryptantha confertiflora, Amsinckia spectabilis, and Amsinckia furcata. The occurrence of heterostyly is well documented in these cases. Interestingly, the anthers of the heterostylous Cryptantha taxa were much larger than those found in other members of this genus. By default, it was assumed that if heterostyly was not specified, the implied condition was to be homostylous. All taxa in Plagiobothrys, Pectocarya, Harpagonella, and the outgroup taxa are homostylous.

Biogeographic Correlation with Morphological Features

Only one morphological trait appears correlated to an ecological trait when examined with the mirror tree approach in Mesquite (Fig. 20). Pollen subshape is variable within Clade II but less so in Clade I (Fig. 15). When compared to the distribution of habitat (dry, moist) on the phylogeny, there appears to be a relationship between taxa that occur in moist habitats in Clade I and the presence of constricted oval circular (coc) pollen. This implies the ancestral pollen condition was coc when members of the subtribe became associated with moist habitats.

This result is confirmed with a 92.54% likelihood of coc being ancestral state of the wet clade (see arrow 2 on Fig. 15). Since this divergence occurred around 5 million years ago at the split between the Miocene and Pleistocene (Fig. 6), insufficient time has passed to allow alternate subshapes to develop. The remainder of the subtribe inhabits drier habitats, and shows a high range of subshape variability. Although Clade I and

Clade II diverged from one another 25-30 million years ago during the Oligocene (Fig.

6), the first taxa in Clade I did not appear until the mid-Miocene. Clade II taxa, however, appeared much earlier - toward the end of the Oligocene (Fig. 6).

Figure 20. Mirror tree (Maddison and Maddison, 2011) of Cryptanthinae with pollen subshape (left) and habitat moisture (right). Subshape abbreviations follow Fig. 15.

Discussion

Cryptanthinae and Geologic Time

The basal node of the Cryptanthinae molecular phylogeny dates back to the mid-

Oligocene (Fig. 6). During this time, global climate was cooling which attributed to reduction of forest biomes and expansion of grassland habitats (Polly et al. 2011). This type of community structure would have been conducive for the evolution of small flowering plants. The Boraginaceae, in addition to many modern angiosperm families flourished during this time (Kazlev 2002). Land mammals dominated Oligocene fauna with an increase in herbivore size as well as micro-mammalian diversity due largely to the adaptation of the ability to digest cellulose (Kazlev 2002; Polly et al 2011). They may have been responsible for the dispersal of many sticky or spikey seeds such as those found in the Cryptanthinae, expanding the range distribution of this subtribe.

During the Oligocene-Miocene boundary, glaciers were advancing (Florindo and

Siegert 2009). Also at this time, Clade II split up into Clade IIA and Clade IIB (Fig. 6,

Fig. 13). Throughout the Miocene, open vegetation systems were favored over closed forests due to an increase in seasonality, reduced rainfall, and an increase in global temperature (Polly et al. 2011). Diversification of Clade II occurred during this time more than 5 million years before the diversification of Clade I. Higher variability in the pollen morphology found in Clade II may be attributed to having more time to accumulate mutations due to desiccation as a primary selection pressure. Interestingly, pollinator diversification resulting in the evolution of modern bee fauna corresponds to the first major adaptive radiation seen in the Cryptanthinae (Patiny 2011).

Another major adaptive radiation occurred in the Cryptanthinae during the

Pliocene. The most noteworthy of these divergences coincides with the wet/dry habitat moisture split (Fig. 18, 20). The climate was cooler and drier than the Miocene, with glacial advances occurring in the late Pliocene and early Pleistocene (Polly et al. 2011)

Also during this time, the Panamanian land bridge formed, uniting North and South

America, and separating the Atlantic and Pacific oceans (Kazlev 2002). This allowed for plant and animal migration, and also correlates with several Cryptanthinae range extensions to South America (Fig. 6, 19).

The Pleistocene is riddled with the most diversification of Cryptanthinae taxa. It is characterized as having more than 20 cycles of glacial advancement and retreat in temperate zones which fragmented many ecosystems (Behrensmeyer 1992). Future work could see if speciation events are correlated with glacial cycles during this epoch.

Noteworthy Cryptanthinae Clades

Historically it has been difficult to identify pollen types beyond the family level in

Angiosperms. This is especially true for Angiosperm families such as the Apiaceae,

Asteraceae, Chenopodiaceae, Cyperaceae, and Poaceae which exhibit very little morphological diversity across genera (Kaltenrieder et al. 2003). However pollen in other plant families, such as the Boraginaceae, show high morphological variability across all genera, making pollen morphology a useful tool for discerning taxonomic relationships in this group.

For example, Amsinckia all share a much larger size (Table 1; Fig. 7; Appendix3), little shape diversity (Fig. 9A, J, Fig. 14; Appendix 3), identical surface textures (Fig. 17;

Appendix 2), and no transverse grooves (Appendix 6) or polar pseudo-apertures

(Appendix 7). They all live in dry habitats (Fig. 18; Appendix 9) and flower in the

Spring (Appendix 10). It is clear in this genus that heterocolpate pollen is the ancestral condition and zonocolpate pollen is derived (Fig. 16). These two clades diverged 5-10 million years ago during the Miocene, and diversified less than 2.6 million years ago during the Pleistocene (Fig. 6). During this time, Earth was warmer than both the

Oligocene and Pliocene. Open vegetation systems such as grasslands, tundra, and deserts were prominent due to drying continental interior, an increase in overall aridity, and an increase in seasonality. The two main Amsinckia lineages persisted through the cooling trend of the Pliocene, and both diversified during the global cooling of the Pleistocene.

Heterostyly arose in Amsinckia spectabilis and A. furcata. This derivation is not unusual for this genus, as the evolution of heterostyly has arisen on multiple occasions, and has been well documented in several species not included in this study (Opler et al.

1975; Olesen 1979; Shoen et al. 1997; Li and Johnston 2001). The parallel evolution of distyly occurs in other Boraginaceae taxa as well (Brys et al. 2008) including Cryptantha flavoculata and C. confertiflora found in this study.

The sister taxa Cryptantha flavoculata and C. confertiflora share many other characteristics (Appendix 2, 3). They are very close in P length (Appendix 11). Both are prolate (Fig. 14), fossulate (Fig. 17), and zonoporate (Fig. 16; Appendix 2, 3) with a transverse groove (Fig. 10; Appendix 6). They live in dry areas (Fig. 18) in North

America (Fig. 19) and flower in the Summer (Appendix 10). Cryptantha flavoculata

41 does not have a polar pseudo-aperture, but interestingly C. confertiflora does (Appendix

3, 7). These two taxa diverged from the rest of Clade IIA during the mid-Miocene, persisted through the Pliocene, and diversified into sister taxa during the Pleistocene (Fig

6). They are more closely related to Pectocarya than the rest of Cryptantha based on molecular evidence (Fig. 6). Hasenstab-Lehman and Simpson (2012) suggested C. confertiflora should be moved to the genus Orecarya based on where it falls out on the phylogeny (Fig. 2). Cryptantha flavoculata was not included in their analysis. Further taxonomic revision is suggested for this clade.

The basal portion of the Amsinckia clade contains three taxa: Plagiobothrys kingii var. harkensii, Pl. kingii var. kingii, and Pl. jonesii (Appendix 2). This clade diverged from Amsinckia 10-15 million years ago during the mid-Miocene (Fig. 6). Pollen morphological characteristics in this group such as size (Table 1; Appendix 11), subshape

(Fig. 15; Appendix 3), and sculpturing (Fig. 17; Appendix 2) are homologous to many of the same characteristics observed in Plagiobothrys. Plagiobothrys jonesii has persisted since the mid-Miocene (Fig. 6). Plagiobothrys kingii var. harkensii and Pl. kingii var. kingii diversified during the transition between the Pleistocene and the Holocene (Fig. 6), an epoch marked by the extinction of many large mammals and the introduction of humans (Polly et al. 2011). Hasenstab-Lehman and Simpson (2012) suggested these taxa should be in the subgenus Amsinckiopsis based on where they fall out in the phylogeny

(Fig. 3). Additional taxonomic revision is suggested for this clade.

Sister taxa Pectocarya anomala and Pe. brachycera are the only two with asymmetrical pollen (Fig. 9; Appendix 2, 3). Samples from a second accession confirmed this trait was fixed, and not just an artifact from the initial collection. This

42 trait, as well as an increased number of pores (which contributes to the deformation of shape) has occasionally been observed in hybrids (Aldridge and Campbell 2006;

Karlsdottir et al. 2008). These sister taxa also share fossulate sculpturing (Fig. 17), heterocolpate pores (Fig. 16; Appendix 2, 3), and no transverse grooves (Appendix 6) or polar pseudo-apertures (Appendix 7). They both live in dry (Fig. 18; Appendix 9), South

American habitats (Fig. 19) and flower in the spring (Appendix 10). Pectocarya anomala pollen is smaller than Pe. brachycera pollen, however, putting Pe. anomala in a different shape class (Fig. 9, 14). These sister taxa diverged from the rest of Clade IIA during the later portion of the Miocene, persisted through the Pliocene, and diversified during the Pleistocene (Fig. 6). This is the first reported case of asymmetrical pollen in the Boraginaceae. Chromosomal analysis for these two taxa is suggested to determine the ploidy level of these two taxa which may be helpful in understanding their unusual shape.

Another group with remarkably similar pollen features in the Cryptanthinae is the clade comprised of Plagiobothrys uncinatus, Pl. verrucosus, and Pl. tenellus (Appendix

2). This clade diverged approximately 15 mya during the mid-Miocene (see arrow 2 on

Fig. 15). Diversification into these taxa started in the late-Pliocene and continued through the Pleistocene (Fig. 6). Pollen in this clade shares the shortest polar length (Fig.

8; Appendix 11), depressed oval subshape (Fig. 9, 15; Appendix 3), fossulate sculpturing

(Fig. 17; Appendix 2), no transverse grooves (Appendix 6), and a presence of polar pseudo-apertures(Appendix 7). They are all found in dry North American habitats (Fig.

18, 19), and flower in the Spring (Appendix 10). During the Pleistocene, Pl. uncinatus derived zonoporate apertures in this clade (Fig. 6, 16).

Plagiobothrys collinus is a polyphyletic group (Fig. 6). Plagiobothrys collinus var. collinus is found in a different portion of the tree than the other Pl. collinus subspecies. Personal communication with M. Guilliams confirmed the identification of the accession was correct. He suggested the Pl. collinus var. collinus sample may have been contaminated. Morphological data could support its position in either current location or in the Pl. collinus ssp. branch. Resampling the molecular data for this taxon is suggested. Also, further taxonomic revision is suggested for Pl. pringlei which is nested amongst Pl. collinus ssp.

Although the collection of taxa used in this study originated in North America, several range extensions occurred to South America, making the Cryptanthinae subtribe amphitropical in distribution (Fig. 19). Within this group, Pectocarya linearis,

Cryptantha maritima, and Amsinckia menziesii are found in both North and South

America (Fig. 19). This characteristic is otherwise known as an amphitropical disjunction. Amphitropical disjunction is found in members of other Boraginaceae genera as well such as Myosotis laxa (Fig. 19), and Tiquilia sp. (Moore et al. 2006). As the South American taxa diversified, and several of them were able to make their way back to North America. These range extensions occurred during several different epochs.

The first North American re-introduction was Pectocarya setosa which happened during the Miocene more than 10 million years ago (Fig. 6). The second taxon to reverse range was Plagiobothrys mollis, occurring during the Pliocene approximately 3 million years ago (Fig. 6). All other range reversals occurred during the Pleistocene (Fig. 6). A likely vector for this type of dispersion pattern is via migratory birds transporting hitchhiker nutlets (Moore et al. 2006).

Conclusion

This study has provided a template for a way that morphological traits can be used in conjunction with molecular-generated phylogenies to enhance our understanding of evolution. The range of morphological variability within the subfamily Cryptanthinae exceeds what is typically observed in many other plant families, and could be useful characteristic for keying plants out in a group that notoriously has little morphological variability. Overlaying pollen morphological characteristics on a phylogenetic tree has added support to many of the molecular clades.

Using biogeographic traits in a phylogenetic context has further elucidated the way environmental conditions translate into the divergence of species. Putting the

Cryptanthinae in the context of geologic timescale helps further elucidate the selection pressures present at the time of diversification in the subtribe. This type of study will be useful for uncovering evolutionary selection pressures that generate the morphological diversity found in other groups.

Further taxonomic revision is suggested for Plagiobothrys kingii var. harkensii,

Pl. kingii var. kingii, Pl. jonesii, Pl. collinus, Cryptantha flavoculata and C. confertiflora.

Chromosomal analysis is suggested for Pectocarya anomala and Pe. brachycera to determine ploidy. Since this study was not all-inclusive and pollen morphological variation was observed at the species level, it would be worthwhile to examine pollen morphology in the remaining Cryptanthinae taxa. Other morphological analyses could include measuring pollen aperture size and texture. Statistical analyses such as testing for phylogenetic signal and pairwise comparisons would further elucidate relationships between morphology, molecular data, biogeography and time.

Literature Cited

Aldridge G, Campbell DR. 2006. Asymmetrical pollen success in Ipomopsis (Polemoniaceae) contact sites. American Journal of Botany. 93:903-909.

Al-Shehbaz IA. 1991. The genera of Boraginaceae in the Southeastern United States. Journal of the Arnold Arboretum. 1:1-170.

[APG III] Angiosperm Phylogeny Group III. updated 2014. [Cited 2014]. Available from: http://www.mobot.org/MOBOT/research/APweb/

Arrigo N, Therrien J, Anderson CL, Windham MD, Haufler CH, Barker MS. 2013. A total evidence approach to understanding phylogenetic relationships and ecological diversity in Selaginella subg. Tetragonostachys. American Journal of Botany. 100:1642- 1682.

Baldwin BG, Goldman D, Keil DJ, Patterson R, Rosatti TJ, Wilken D, editors. 2012. The Jepson manual higher plants of California. 2nd edition. University of California Press.

Behrensmeyer AK, Damuth JD, DiMichele A, Potts R, Sues HD, Wing SL. 1992. Terrestrial ecosystems through time: evolutionary paleoecology of terrestrial plants and animals. Chicago. University of Chicago Press.

Bentham G, Hooker JD. 1873. Genera plantarum :ad exemplaria imprimis in herberiis kewensibus servata definite. Lovell & Reeve Company.

Biznet R, Kandemir I, Orcan N. 2010. Palynological classification of Onosma L. (Boraginaceae) species from east Mediterranean region in Turkey. Acta Botanica Croatia. 69:259-274.

Brys R, Jacquemyn H, Hermy M, Beckman T. 2008. Pollen deposition rates and the functioning of distyly in the perennial Pulmonaria officinalis (Boraginaceae). Plant Systematic Evolution. 273:1-12.

Buys MH, Hilger HH. 2003. Boraginaceae cymes are exclusively scorpioid and not helicoid. Taxon. 52:719-724.

Cohen J. 2011. A phylogenetic analysis of morphological and molecular characters of Lithospermum L. (Boraginaceae) and related taxa: evolutionary relationships and character evolution. Cladistics. 27:559-580.

[CCH] Consortium of California Herbaria. Regents of the University of California. 2010. [Cited 2014]. Available from: http://ucjeps.berkeley.edu/consortium/

Darwin C. 1877. The different forms of flowers on plants of the same species. London: John Murray.

Davis, O. 2001. University of Arizona catalog of internet pollen and spore images. [cited 2014]. Available from: http://www.geo.arizona.edu/palynology/polonweb.html

Diez MJ, Valdez B. 1991. Pollen morphology of the tribes Eritrichieae and Cynoglosseae (Boraginaceae) in the Iberian Peninsula and its taxonomic significance. Botanical Journal of the Linnean Society. 107:49-66.

Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology. 7:214.

Eernisse DJ, Kluge AG. 1993. Taxonomic congruence versus total evidence, and amniote phylogeny inferred from fossils, molecules, and morphology. Molecular Biology and Evolution. 10:1170-1195.

Erdtman G. 1966. Pollen morphology and plant taxonomy. Hafner Publishing Company.

Faegri K, Iversen J. 1975. Textbook of pollen analysis. Hafner Press.

Falatoury AN, Pakravan M, Sharifnia F. 2011. Palynological study of Nonea (Boraginaceae-Boragineae) in Iran. Progress in Biological Sciences. 1:36-43.

Florindo F, Siegert M, editors. 2009. Developments in Earth and environmental sciences. Volume 8. Elsevier B.V.

Furness CA, Rudall PJ. 2004. Pollen aperture evolution – a crucial factor for eudicot success?. TRENDS in Plant Science. 9:154-158.

Fukuda T, Ikeda H. 2012. Palynological analysis and taxonomic position of the genus Mertensia (Boragincaeae). Botany. 90:722-730.

Galarini R, Ricciardelli D’Albore M, editors. 1998. Mediteranean melissopalynology. [Cited 2014]. Available from: http://www.izsum.it/Melissopalynology/index.htm?8

Grimm D, Almeda F. 2013. Systematics, phylogeny, and biogeography of Chaetolepis (Melastomataceae). Journal of the Botanical Research Institute of Texas. 7:217-263.

Gottschling M, Hilger HH, Diane N. 2001. Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny Boraginales. Plant Biology. 3:629-636.

Guilliams M. 2013. Cryptanthinae Molecular Phylogeny. University of California at Berkeley. Unpublished data.

Gürke M, Engler A, Prantl K, editors. 1897. Boraginaceae. Die Natürlichen Pflanzenfamilien. Engelmann, Leipzig. 4:71-131.

Hargrove L, Simpson MG. 2003. Ultrastructure of heterocolpate pollen in Cryptantha (Boraginaceae). International Journal of Plant Science. 164:137-151.

Hasenstab-Lehman KE, Simpson MG. 2012. Cat’s eyes and popcorn flowers: Phylogenetic systematics of the genus Cryptantha s.l. (Boraginaceae). Systematic Botany. 37:738-757.

Hilger H, Selvi F, Papini A, Bigazzi M. 2004. Molecular systematics of Boraginaceae tribe Boragineae based on ITS1 and trnL sequences, with special reference to Anchusa s.l. Annals of Botany. 94:201-212.

Horn N. 2000. Revision der gattungen Plagiobothrys and Pectocarya in Chile und den angrenzenden gebieten. Universität München. Maximilians.

Huang J, Zhang M, Cohen JI. 2013. Phylogenetic analysis of Lappula Moench (Boraginaceae) based on molecular and morphological data. Plant Systematic Evolution. 299:913-926.

Huggins PM, Li W, Haws D, Friedrich T, Liu J, Yoshida R. 2011. Bayes estimators for phylogenetic reconstruction. Systematic Biology. 60:428-540.

Jones G, E.F.Wilson EF. 1995. Pollen as indicators of source areas and foraging resources. [APMRU] Areawide Pest Management Research Unit, USDA. [cited 2012] Available from: http://pollen.usda.gov/index.htm

Kaltenrieder P, von Ballmoos P, Hoen P editor. 2003. Introduction to pollen analysis. [cited 2014]. Available from: http://www.botany.unibe.ch/paleo/pollen_e/index.htm

Kapp RO 1969. How to know pollen and spores. W.M.C. Brown Company Publishers.

Karlsdottir L, Hallsdottir M, Thorsson AT, Anamthawat-Jonsson K. 2008. Characteristics of pollen from natural triploid Betula hybrids. Grana. 47:52-59.

Kay K, Whittall JB, Hodges SA. 2006. A survey of nuclear ribosomal internal transcribed spacer substitution rates across angiosperms: an approximate molecular clock with life history effects. BMC Evolutionary Biology. 6:36.

Kazlev, MA. 2002. Palaeos: The Oligocene. [cited 2014]. Available from: http://palaeos.com/cenozoic/oligocene/oligocene.htm

Langstrom E, Chase MW. 2002. Tribes of the Boraginoideae (Boraginaceae) and placement of Echiochilon, Ogastemma, and Sericostoma: a phylogenetic analysis based on atpB plastid DNA sequence data. Plant Systematic Evolution. 234:137-153.

Lazarova M, Koutsios A, Kontopoulos N. 2012. Holocene vegetation history of the Kotihi lagoon (northwest Peloponnesus, Greece). Quaternary International. 261:138- 145.

Lee MSY, Camens AB. 2009. Strong morphological support for the molecular evolutionary tree of placental mammals. Journal of Evolutionary Biology. 22:2243- 2257.

Li P, Johnston MO. 2001. Comparative floral morphometrics of distyly and homostyly in three evolutionary lineages of Amsinckia (Boraginaceae). Canadian Journal of Botany. 79:1332-1348.

Lu L, Wang W, Chen Z, Wen J. 2013. Phylogeny of the non-monophyletic Cayratia Juss. (Vitaceae) and implications for character evolution and biogeography. Molecular Phylogenetics and Evolution. 68:502-515.

Maddison WP, Maddison DR. 2011. Mesquite: a modular system for evolutionary analysis. Version 2.75. [cited 2012]. Available from: http://mesquiteproject.org

Martin PS, Drew CM. 1969. Scanning electron photomicrographs of southwestern pollen grains. Journal Arizona Academy of Sciences. 5:147-176.

Moore MJ, Tye A, Jansen RK. 2006. Patterns of long-distance dispersal in Tiquilia subg. Tiquilia (Boraginaceae): Implications for the origins of amphitropical disjuncts and Galapagos Islands endemics. American Journal of Botany. 93:1163-1177.

Mozaffar MK, Osaloo AK, Oskoueyan R, Saffar KN, Amirahmadi A. 2013. Tribe Eritrichieae (Boraginaceae s.str.) in West Asia: a molecular phylogenetic perspective. Plant Systematic Evolution. 299:197-208.

Naik VN. 1984. Taxonomy of angiosperms. McGraw Hill Education.

Nazaire M, Hufford L. 2012. A broad phylogenetic analysis of Boraginaceae: Implications for the relationships of Mertensia. Systematic Botany. 37:758-783.

Olesen JM. 1979. Floral morphology and pollen flow in the heterostylous species Pulmonaria obscura Dumort (Boraginaceae). New Phytology. 82:757-767.

Olmstead RG. 2013. Phylogeny and biogeography in Solanaceae, Verbenaceae, and Bignoniaceae: a comparison of continental and intercontinental diversification patterns. Botanical Journal of the Linnean Society. 171:80-102.

Opler PA, Baker HG, Frankie GW. 1975. Reproductive biology of some Costa Rican Cordia species (Boraginaceae). Biotropica. 7:234-247.

Ortega-Rosas CI, Guiot J, Penalba MC, Ortiz-Acosta ME. 2008. Biomization and quantitative climate reconstruction techniques in northwestern Mexico – with an application to four Holocene pollen sequences. Global and Planetary Change. 61:242- 266.

Patiny, S, editor. 2011. Evolution of plant-pollinator relationships. Cambridge University Press.

Polly D, Guralnick R, Collins A, Speer B, Rieboldt S, Smith D, editors. The Cenozoic Era. 2011. [cited 2014]. Available from: http://www.ucmp.berkeley.edu/cenozoic/cenozoic.php

Punt W, Hoen PP, Blackmore S, Nilsson S, LeThomas A. 2007. Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology. 143:1-81.

Rambaut, A. 2012. FigTree v1.4. [cited 2013]. Available from: http://tree.bio.ed.ac.uk/software/figtree/

Scheel R, Ybert JP, Barth OM. 1966. Pollen morphology of the Boraginaceae from Santa Catarina State (southern Brazil), with comments on the taxonomy of the family. Grana. 35:138-153.

Schluter D, Price T, Mooers A, Ludwig D. 1997. Likelihood of ancestor states in adaptive radiation. Evolution. 51:1699-1711.

Schwartzer C. 2007. Systematische untersuchungen an den peruanischen vertretern der gattungen Pectocarya D.C. ex Meisn., Amsinckia Lehm., Plagiobothrys Fisch. und C.A.Mey. und Cryptantha Lehm. ex G.Don (Boraginaceae). Berlin. Freie University.

Shoen DJ, Johnston MO, L’Heureux AM, Marsolais JV. 1997. Evolutionary history of the mating system in Amsinckia (Boraginaceae). Evolution. 51:1090-1099.

Simpson M. 2006. Plant systematics. Elsevier Academic Press.

Singh G. 2004. Plant systematics: an integrated approach. Science Publishers, Inc.

Solomon AM, King JE, Martin PS, Thomas J. 1974. Further scanning electron photomicrographs of southwestern pollen grains. Journal Arizona Academy of Sciences. 8:135-137.

Tai SS, Tang XM. 2001. Manipulating Biological Samples for Environmental Scanning Electron Microscopy Observation. Scanning. 23:267-272.

Takhtajan A. 1997. Diversity and classification of flowering plants. Columbia University Press.

Volkova OA, Severova EE, Polevova SV. 2013. Structural basis of harmomegathy: evidence from Boraginaceae pollen. Plant Systematic Evolution. 299:1769-1779.

Watson L, Dallwitz MJ. 1992 onwards. The families of flowering plants: descriptions, illustrations, identification, and information retrieval. Version: 22nd April 2014. [cited 2014]. Available from: http://delta-intkey.com’

Weigend W, Gottschling M, Selvi F, Hilger HH. 2010. Fossil and extant western hemisphere Boragieae, and the polyphyly of Trigonotideae Riedl (Boraginaceae: Boraginoideae). Systematic Botany. 35:409-419.

Xu B, Gao XF, Wu N, Zhang LB. 2011. Pollen diversity and its systematic implications in Lespedeza (Fabaceae). Systematic Botany. 36:352-361.

Zheng L, Yang J, Chen X. 2012. Phylogeny of the Labeoninae (Teleostei, Cypriniformes) based on nuclear DNA sequences and implications on character evolution and biogeography. Current Zoology. 58:837-850.

Zomlefer WB. 1994. Guide to flowering plant families. The University of North Carolina Press Chapel Hill and London.

Appendix 1. Table 2. Cryptanthinae taxa sampled with Herbarium and accession information. Abbreviations are as follows: NCC - North Coast California, UC/JEPS – University of California at Berkeley/Jepson, UAZ – University of Arizona, CONC – Universidad de Concepción Peru, MO – Missouri Botanical Gardens.

Taxon Herbarium Accession # Amsinckia eastwodiae J.F. MacBr. NCC 19882 Amsinckia furcata Suksd. NCC 21214 Amsinckia menziesii (Lehm.) Nelson & J.F. Macbr. var. intermedia UC/JEPS M191034 Fisch. & C.A. Mey Amsinckia menziesii (Lehm.) Nelson & J.F. Macbr. var. menziesii UC/JEPS M178324 Amsinckia retrorsa Suksd. NCC 1604 Amsinckia spectabilis Fischer & C. Meyer NCC 1614 Amsinckia vernicosa Hook. & Arn UC/JEPS 1573179 Cryptantha circumscissa (Hook. & Arn.) I.M. Johnston NCC 1666 Cryptantha confertiflora (E. Greene) Payson NCC 1678 Cryptantha costata Brandegee UC/JEPS 67548 Cryptantha flaccida (Lehm.) E. Greene NCC 24774 Cryptantha flavoculata (Nelson) Payson UC/JEPS 90302 Cryptantha gracilis Osterhout NCC 1706 Cryptantha maritima (E. Greene) E. Greene NCC 1763 Cryptantha micromeres (A. Gray) E. Greene UC/JEPS 84746 Cryptantha muricata (Hook. & Arn.) Nelson & J.F. Macbr NCC 1797 Cryptantha racemosa (S. Watson) E. Greene UC/JEPS 1534924 Cryptantha simulans E. Greene NCC 1824 Harpagonella palmeri A. Gray var. arizonica I.M. Johnston UAZ 94692 Harpagonella palmeri A. Gray var. palmeri UC/JEPS 67974 Myosotis discolor Pers. NCC 2268 Myosotis laxa Lehm. NCC 21645 Pectocarya anomala I.M. Johnston CONC 27476 Pectocarya brachycera Sp. Nov. Ined. CONC 140095 Pectocarya dimorpha I.M. Johnston CONC 143358 Pectocarya linearis (Ruiz & Pavón) DC. CONC 166948 Pectocarya penicillata (Hook. & Arn.) A. DC NCC 1961 Pectocarya platycarpa (Munz & I.M. Johnston) Munz & I.M. UC/JEPS 23033 Johnston Pectocarya pusilla (A. DC.) A. Gray NCC 21278 Pectocarya setosa A. Gray NCC 1986 Plagiobothrys acanthocarpus (Piper) I.M. Johnston NCC 19880 Plagiobothrys albiflorus R.L. Pérez-Mor. MO 2386108 Plagiobothrys arizonicus (A. Gray) A. Gray NCC 1989

Taxon (cont.) Herbarium Accession # Plagiobothrys austinae (E. Greene) I.M. Johnston UC/JEPS 60925 Plagiobothrys bracteatus (T.J. Howell) I.M. Johnston NCC 21699 Plagiobothrys calandrinoides (Phil.) I.M.Johnston MO 1657468 Plagiobothrys canescens Benth. var. canescens UC/JEPS 104459 Plagiobothrys canescens Benth. var. catalinensis (A. Gray) Jeps. UC/JEPS 117191 Plagiobothrys cognatus (E. Greene) I.M. Johnston NCC 2043 Plagiobothrys collinus (Phil.) I.M. Johnston var. californicus (A. NCC 21455 Gray) Higgins Plagiobothrys collinus (Phil.) I.M. Johnston var. collinus CONC 112974 Plagiobothrys collinus (Phil.) I.M. Johnston var. fulvescens (I.M. UC/JEPS 1928510 Johnston) Higgins Plagiobothrys collinus (Phil.) I.M. Johnston var. gracilis (I.M. UC/JEPS 1787352 Johnston) Higgins Plagiobothrys collinus (Phil.) I.M. Johnston var. ursinus (A. Gray) UC/JEPS 20150 Higgins Plagiobothrys congestus (Wedd.) I.M.Johnston MO 4966800 Plagiobothrys corymbosus (Ruiz et. Pav.) I.M. Johnston CONC 42058 Plagiobothrys cusickii (E. Greene) I.M. Johnston UC/JEPS 1191841 Plagiobothrys diffusus (Greene) Jtn. UC/JEPS 517409 Plagiobothrys distantiflorus (Piper) M. Peck UC/JEPS 882337 Plagiobothrys figuratus (Piper) IM Johnston ex M. Peck UC/JEPS M180478 Plagiobothrys fulvus (Hook. & Arn.) I.M. Johnston var. fulvus MO 116066 Plagiobothrys glyptocarpus (Piper) I.M. Johnston var. glyptocarpus UC/JEPS 1071083 Plagiobothrys glyptocarpus (Piper) I.M. Johnston var. modestus I.M. UC/JEPS 1347183 Johnston Plagiobothrys greenei (A. Gray) I.M. Johnston NCC 23635 Plagiobothrys hispidulus (E. Greene) I.M. Johnston NCC 2067 Plagiobothrys humilis (Ruiz & Pav.) I.M. Johnston MO 965459 Plagiobothrys humistratus (E. Greene) I.M. Johnston UC/JEPS 765636 Plagiobothrys infectivus I.M. Johnston UC/JEPS M285288 Plagiobothrys jonesii A. Gray UC/JEPS 666216 Plagiobothrys kingii (S. Watson) A. Gray var. harkensii (E. Greene) UC/JEPS 882338 Jepson Plagiobothrys kingii (S. Watson) A. Gray var. kingii UC/JEPS 1483821 Plagiobothrys kunthii (Walp.) I.M. Johnston MO 4265995 Plagiobothrys linifolius (Lehm.) I.M. Johnston MO 3771318 Plagiobothrys mollis (A. Gray) I.M. Johnston var. mollis UC/JEPS 1587084 Plagiobothrys parishii I.M. Johnston UC/JEPS 184494 Plagiobothrys pringlei Greene UC/JEPS 126033 Plagiobothrys procumbens (Colla) Colla MO 1617599 Plagiobothrys scouleri (Hook. & Arn.) I.M. Johnston UC/JEPS M222205

Taxon (cont.) Herbarium Accession # Plagiobothrys scriptus (E. Greene) I.M. Johnston UC/JEPS M284988 Plagiobothrys strictus (E. Greene) I.M. Johnston NCC 2114 Plagiobothrys tenellus (Nutt.) A. Gray UC/JEPS 1871083 Plagiobothrys tener (Greene) I.M. Johnston var. tener UC/JEPS 726338 Plagiobothrys uliginosus I.M. Johnston MO 826312 Plagiobothrys uncinatus J. Howell UC/JEPS M212084 Plagiobothrys undulatus (Piper) I.M. Johnston NCC 23640 Plagiobothrys verrucosus (Phil.) I.M. Johnston MO 1166524

Appendix 2. Figure 21. A selection of Scanning Electron Microscope images of Cryptanthinae pollen taken for this study. Pollen shown in each image may not necessarily be the Archetype, but instead are part of the natural size variation within each taxon. Images are listed alphabetically. Scale bars vary. Legend for SEM images is shown below.

Amsinckia intermedia Fisch. & C.A. Mey

Amsinckia eastwodiae J.F. MacBr. Amsinckia menziesii (Lehm.) Nelson & J.F. Macbr.

Amsinckia furcata Suksd. Amsinckia retrorsa Suksd.

Amsinckia spectabilis Fischer & C. Meyer Cryptantha confertiflora (E. Greene) Payson

Amsinckia vernicosa Hook. & Arn Cryptantha costata Brandegee

Cryptantha circumscissa (Hook. & Arn.) Cryptantha flaccida (Lehm.) E. Greene I.M. Johnston

Cryptantha flavoculata (Nelson) Payson Cryptantha micromeres (A. Gray) E. Greene

Cryptantha gracilis Osterhout Cryptantha muricata (Hook. & Arn.) Nelson & J.F. Macbr

Cryptantha maritima (E. Greene) E. Cryptantha racemosa (S. Watson) E. Greene Greene

Cryptantha simulans E. Greene Myosotis discolor Pers.

Harpagonella palmeri A. Gray var. Myosotis laxa Lehm. arizonica I.M. Johnston

Harpagonella palmeri A. Gray var. palmeri Pectocarya anomala I.M. Johnston

Pectocarya brachycera Sp. Nov. Ined. Pectocarya penicillata (Hook. & Arn.) A. DC

Pectocarya dimorpha I.M. Johnston Pectocarya platycarpa (Munz & I.M. Johnston) Munz & I.M. Johnston

Pectocarya linearis (Ruiz & Pavón) DC. Pectocarya pusilla (A. DC.) A. Gray

Pectocarya setosa A. Gray Plagiobothrys arizonicus (A. Gray) A. Gray

Plagiobothrys acanthocarpus (Piper) I.M. Plagiobothrys austinae (E. Greene) I.M. Johnston Johnston

Plagiobothrys albiflorus R.L. Pérez-Mor. Plagiobothrys bracteatus (T.J. Howell) I.M. Johnston

Plagiobothrys calandrinoides (Phil.) Plagiobothrys cognatus (E. Greene) I.M. I.M.Johnston Johnston

Plagiobothrys canescens Benth. var. Plagiobothrys collinus (Phil.) I.M. canescens Johnston var. californicus (A. Gray) Higgins

Plagiobothrys canescens Benth. var. Plagiobothrys collinus (Phil.) I.M. catalinensis (A. Gray) Jeps. Johnston var. collinus

Plagiobothrys collinus (Phil.) I.M. Plagiobothrys congestus (Wedd.) Johnston var. fulvescens (I.M. Johnston) I.M.Johnston Higgins

Plagiobothrys collinus (Phil.) I.M. Plagiobothrys corymbosus (Ruiz etPav.) Johnston var. gracilis (I.M. Johnston) I.M. Johnston Higgins

Plagiobothrys collinus (Phil.) I.M. Plagiobothrys cusickii (E. Greene) I.M. Johnston var. ursinus (A. Gray) Higgins Johnston

Plagiobothrys diffusus (Greene) Jtn. Plagiobothrys fulvus (Hook. & Arn.) I.M. Johnston var. fulvus

Plagiobothrys distantiflorus (Piper) M. Plagiobothrys glyptocarpus (Piper) I.M. Peck Johnston var. glyptocarpus

Plagiobothrys figuratus (Piper) IM Plagiobothrys glyptocarpus (Piper) I.M. Johnston ex M. Peck Johnston var. modestus I.M. Johnston

Plagiobothrys greenei (A. Gray) I.M. Plagiobothrys humistratus (E. Greene) I.M. Johnston Johnston

Plagiobothrys hispidulus (E. Greene) I.M. Plagiobothrys infectivus I.M. Johnston Johnston

Plagiobothrys humilis (Ruiz & Pav.) I.M. Plagiobothrys jonesii A. Gray Johnston

Plagiobothrys kingii (S. Watson) A. Gray Plagiobothrys linifolius (Lehm.) I.M. var. harkensii (E. Greene) Jepson Johnston

Plagiobothrys kingii (S. Watson) A. Gray Plagiobothrys mollis (A. Gray) I.M. var. kingii Johnston var. mollis

Plagiobothrys kunthii (Walp.) I.M. Plagiobothrys parishii I.M. Johnston Johnston

Plagiobothrys pringlei Greene Plagiobothrys scriptus (E. Greene) I.M. Johnston

Plagiobothrys procumbens (Colla) Colla Plagiobothrys strictus (E. Greene) I.M. Johnston

Plagiobothrys scouleri (Hook. & Arn.) I.M. Plagiobothrys tenellus (Nutt.) A. Gray Johnston

Plagiobothrys tener (Greene) I.M. Johnston Plagiobothrys undulatus (Piper) I.M. var. tener Johnston

Plagiobothrys uliginosus I.M. Johnston Plagiobothrys verrucosus (Phil.) I.M. Johnston

Plagiobothrys uncinatus J. Howell

Appendix 3. Figure 22. Line drawings (equatorial view left, polar view right) generated for Cryptanthinae pollen archetypes based on the measurements shown in Fig. 4. Name of taxon, P:E ratio, and subshape are listed for each drawing. Abbreviations for genera are: Pl. – Plagiobothrys; Pe. – Pectocarya; H. – Harpagonella; C. – Cryptantha; A. – Amsinckia; M. – Myosotis. Abbreviations for subshapes are: co - compressed oval, coc - constricted oval circular, cr - compressed rectangular, o - oval, do - depressed oval, b - biconvex, rh - rhomboidal. Key for figures is listed below.

Exine deposits which dileneate true apertures from pseudo-apertures.

Pseudo-apertures with texture that differes from true apertures

Texture around apertures

Echinate sculpturing

Fossulate sculpturing

Foveolate sculpturing

Gemmate-clavate sculpturing

Appendix 4. Table 3. Pollen shape and subshape distribution among 74 taxa in the Cryptanthinae. The taxa are organized with regard to Erdtman (1966) shape as well as Faegri and Iversen (1975) subshape. The three different shapes are based on P:E ratios. The subshapes are based on the measurements taken in Fig. 4. The Myosotis outgroup species are included as well.

Shape Subshape Taxa Subprolate (4 taxa) oval A menziesii depressed oval Pl uncinatus, Pl tenellus biconvex Pe anomala Prolate (29 taxa) oval Pe setosa rhomboidal A eastwoodiae, A furcata, A intermedia, A vernicosa constricted oval circular C confertiflora, M laxa, Pe linearis, Pe platycarpa, Pl collinus v collinus, Pl collinus v ursinus, Pl glyptocarpus v modestus, Pl humistratus, Pl kingii v kingii

constricted rectangular Pl fulvus v campestris, Pl humilis compressed oval C flavoculata, C gracilis, M discolor, Pe penicillata, Pl jonesii, Pl infectivus depressed oval A retrorsa, A spectabilis, H palmeri v arizonica, Pe pusilla, Pl verrucosus biconvex Pe brachycera Perprolate (43 taxa) constricted oval circular C circumscissa, C costata, C flaccida, C maritima, C muricata, C racemosa, Pl albiflorus, Pl austinae, Pl bracteatus, Pl calandrinoides, Pl canescens v canescens, Pl canescens v catilinense, Pl cognatus, Pl collinus v californicus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl congestus, Pl corymbosus, Pl cusickii, Pl diffusus, Pl dististantiflorus, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl greenei, Pl hispidus, Pl kingii v harkensii, Pl kunthii, Pl linifolius, Pl mollis v mollis, Pl parishii, Pl scouleri, Pl scriptus, Pl strictus, Pl tener v tener, Pl uliginosus, Pl undulates compressed oval C simulans, H palmeri v palmeri, Pe dimorpha, Pl acanthocarpus, Pl arizonicus, Pl pringlei, Pl procumbens

Appendix 5. Table 4. Pollen aperture types found across 74 taxa of the Cryptanthinae. The Myosotis outgroup species are included as well.

Aperture Type Taxa Heterocolpate A furcata, A vernicosa, C circumscissa, C costata, C flaccida, C gracilis, C maritima, C (63 taxa) micromeres, C muricata, C racemosa, C simulans, H palmeri v arizonica, M discolor, M laxa, Pe anomala, Pe brachycera, Pe dimorpha, Pe linearis v ferocula, Pe platycarpa, Pe pusilla, Pe setosa, Pl acanthocarpus, Pl arizonicus, Pl austinae, Pl bracteatus, Pl calandrinoides, Pl canescens v canescens, Pl canescens v catilenense, Pl cognatus, Pl collinus v californicus, Pl collinus v collinus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl collinus v ursinus, Pl congestus, Pl corymbosus, Pl cusickii, Pl diffusus, Pl distantiflorus, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl greenei, Pl hispidulus, Pl humilis, Pl humistratus, Pl infectivus, Pl jonesii, Pl kingii v harkensii, Pl kingii v kingii, Pl kunthii, Pl linifolius, Pl mollis v mollis, Pl parishii, Pl pringlei, Pl procumbens, Pl scouleri, Pl scriptus, Pl strictus, Pl tenellus, Pl tener v tener, Pl uliginosus, Pl undulatus, Pl verrucosus

Zonocolpate A eastwoodiae, A intermedia, A menziesii, A retrorsa, A spectabilis, H palmeri v palmeri, (8 taxa) Pl albiflorus, Pl glyptocarpus v modestus

Zonoporate C confertiflora, C flavoculata, Pe penicillata, Pl fulvus v campestris, Pl uncinatus (5 taxa)

Appendix 6. Table 5. Presence or absence of transverse grooves in 74 taxa of Cryptanthinae pollen. The Myosotis outgroup species are included as well.

Transverse groove Taxa Absent A eastwoodiae, A furcata, A intermedia, A menziesii, A retrorsa, A spectabilis, A (67 taxa) vernicosa, C circumscissa, C costata, C flaccida, C gracilis, C maritima, C micromeres, C muricata, C racemosa, C simulans, M discolor, M laxa, Pe anomala, Pe brachycera, Pe linearis v ferocula, Pe platycarpa, Pe pusilla, Pe setosa, Pl acanthocarpus, Pl albiflorus, Pl arizonicus, Pl austinae, Pl bracteatus, Pl calandrinoides, Pl canescens v canescens, Pl canescens v catilenense, Pl cognatus, Pl collinus v californicus, Pl collinus v collinus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl collinus v ursinus, Pl corymbosus, Pl cusickii, Pl diffusus, Pl distantiflorus, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl glyptocarpus v modestus, Pl greenei, Pl humilis, Pl humistratus, Pl infectivus, Pl jonesii, Pl kingii v harkensii, Pl kingii v kingii, Pl kunthii, Pl linifolius, Pl mollis v mollis, Pl parishii, Pl pringlei, Pl procumbens, Pl scouleri, Pl scriptus, Pl strictus, Pl tenellus, Pl tener v tener, Pl uliginosus, Pl undulatus, Pl verrucosus

Present C confertiflora, C flavoculata, H palmeri v arizonica, H palmeri v palmeri, Pe (9 taxa) dimorpha, Pe penicillata, Pl congestus, Pl fulvus v campestris, Pl hispidulus

Appendix 7. Table 6. Presence or absence of polar pseudo-apertures across 74 taxa of Cryptanthinae pollen. The Myosotis outgroup species are included as well.

Polar Pseudo-apertures Taxa Absent A eastwoodiae, A furcata, A intermedia, A menziesii, A retrorsa, A (43 taxa) spectabilis, A vernicosa, C costata, C flavoculata, C maritima, C micromeres, C racemosa, C simulans, H palmeri v arizonica, H palmeri v palmeri, M laxa, Pe anomala, Pe brachycera, Pe dimorpha, Pe pusilla, Pl acanthocarpus, Pl albiflorus, Pl calandrinoides, Pl canescens v canescens, Pl collinus v collinus, Pl collinus v ursinus, Pl congestus, Pl cusickii, Pl fulvus v campestris, Pl glyptocarpus v modestus, Pl greenei, Pl hispidulus, Pl humistratus, Pl jonesii, Pl kingii v harkensii, Pl kingii v kingii, Pl linifolius, Pl pringlei, Pl procumbens, Pl scriptus, Pl uliginosus, Pl undulatus Present C circumscissa, C confertiflora, C flaccida, C gracilis, M discolor, Pe (34 taxa) linearis v ferocula, Pe penicillata, Pe platycarpa, Pe setosa, Pl arizonicus, Pl austinae, Pl bracteatus, Pl canescens v catilenense, Pl cognatus, Pl collinus v californicus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl corymbosus, Pl diffusus, Pl distantiflorus, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl humilis, Pl infectivus, Pl kunthii, Pl mollis v mollis, Pl parishii, Pl scouleri, Pl strictus, Pl tenellus, Pl tener v tener, Pl uncinatus, Pl verrucosus

Appendix 8. Table 7. Sculpturing types for pollen of 74 taxa in the Cryptanthinae. The Myosotis outgroup species are included as well.

Sculpturing Taxa Echinate Pe platycarpa (1 taxon) Fossulate C circumscissa, C confertiflora, C flaccida, C flavoculata, C maritima, C muricata, C (54 taxa) racemosa, C simulans, M discolor, Pe anomala, Pe brachycera, Pe dimorpha, Pe penicillata, Pe pusilla, Pe setosa, Pl albiflorus, Pl bracteatus, Pl calandrinoides, Pl canescens v canescens, Pl canescens v catilenense, Pl cognatus, Pl collinus v californicus, Pl collinus v collinus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl collinus v ursinus, Pl congestus, Pl corymbosus, Pl cusickii, Pl diffusus, Pl distantiflorus, Pl figuratus, Pl fulvus v campestris, Pl glyptocarpus v glyptocarpus, Pl glyptocarpus v modestus, Pl hispidulus, Pl humilis, Pl humistratus, Pl infectivus, Pl jonesii, Pl kingii v harkensii, Pl kingii v kingii, Pl kunthii, Pl linifolius, Pl mollis v mollis, Pl parishii, Pl scouleri, Pl scriptus, Pl tenellus, Pl tener v tener, Pl uncinatus, Pl undulatus, Pl verrucosus

Foveolate C costata, H palmeri v arizonica, H palmeri v palmeri, M laxa, Pe linearis v ferocula, (12 taxa) Pl acanthocarpus, Pl arizonicus, Pl greenei, Pl pringlei, Pl procumbens, Pl strictus, Pl uliginosus

Gemmate clavate C gracilis, C micromeres (2 taxa) Reticulate A eastwoodiae, A furcata, A intermedia, A menziesii, A retrorsa, A spectabilis, A (7 taxa) vernicosa

Appendix 9. Table 8. Habitat moisture level for 74 Cryptanthinae taxa and two Myosotis outgroup taxa.

Habitat Moisture Taxa Dry A eastwoodiae, A furcata, A intermedia, A menziesii, A retrorsa, A spectabilis, A (44 taxa) vernicosa, C circumscissa, C confertiflora, C costata, C flaccida, C flavoculata, C gracilis, C maritima, C micromeres, C muricata, C racemosa, C simulans, H palmeri v arizonica, H palmeri v palmeri, Pe anomala, Pe brachycera, Pe dimorpha, Pe linearis v ferocula, Pe penicillata, Pe platycarpa, Pe pusilla, Pe setosa, Pl jonesii, Pl arizonicus, Pl canescens v canescens, Pl canescens v catilenense, Pl collinus v californicus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl collinus v ursinus, Pl fulvus v campestris, Pl infectivus, Pl kingii v harkensii, Pl kingii v kingii, Pl pringlei, Pl tenellus, Pl uncinatus, Pl verrucosus

Wet M discolor, M laxa, Pl acanthocarpus, Pl albiflorus, Pl austinae, Pl bracteatus, Pl (32 taxa) calandrinoides, Pl cognatus, Pl collinus v collinus, Pl congestus, Pl corymbosus, Pl cusickii, Pl diffusus, Pl distantiflorus, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl glyptocarpus v modestus, Pl greenei, Pl hispidulus, Pl humilis, Pl humistratus, Pl kunthii, Pl linifolius, Pl mollis v mollis, Pl parishii, Pl procumbens, Pl scouleri, Pl scriptus, Pl strictus, Pl tener v tener, Pl uliginosus, Pl undulatus

Appendix 10. Table 9. Flowering period in selected Cryptanthinae taxa.

Flowering Period Taxa Spring A eastwoodiae, A furcata, A intermedia, A menziesii, A retrorsa, A spectabilis, A (51 taxa) vernicosa, C costata, C flaccida, C maritima, C micromeres, C muricata, C racemosa, H palmeri v arizonica, H palmeri v palmeri, Pe anomala, Pe brachycera, Pe dimorpha, Pe linearis v ferocula, Pe penicillata, Pe platycarpa, Pe pusilla, Pe setosa, Pl acanthocarpus, Pl arizonicus, Pl austinae, Pl bracteatus, Pl canescens v canescens, Pl canescens v catilenense, Pl collinus v californicus, Pl collinus v collinus, Pl collinus v fulvescens, Pl collinus v gracilis, Pl diffusus, Pl fulvus v campestris, Pl glyptocarpus v modestus, Pl greenei, Pl humistratus, Pl infectivus, Pl jonesii, Pl kingii v harkensii, Pl linifolius, Pl pringlei, Pl scriptus, Pl strictus, Pl tenellus, Pl uliginosus, Pl uncinatus, Pl undulatus, Pl verrucosus

Summer C circumscissa, C confertiflora, C flavoculata, C gracilis, C simulans, M (25 taxa) discolor, M laxa, Pl albiflorus, Pl calandrinoides, Pl cognatus, Pl collinus v ursinus, Pl congestus, Pl corymbosus, Pl cusickii, Pl figuratus, Pl glyptocarpus v glyptocarpus, Pl hispidulus, Pl humilis, Pl kingii v kingii, Pl kunthii, Pl mollis v mollis, Pl parishii, Pl procumbens, Pl scouleri, Pl tener v tener

Appendix 11. Table 10. Average polar and equatorial diameter, P:E ratio, standard deviation of P and E. A standard test of normality for both P and E was performed in excel and all were normal with a significance of p<.05. Taxa P E P:E stdev P stdev E Pl. parishii 5.516 2.157 2.56 0.207 0.201 Pl. bracteatus 7.912 3.893 2.03 0.454 0.366 Pl. glyptocarpus v modestus 7.682 3.963 1.94 0.766 0.550 Pl. hispidulus 5.666 1.681 3.37 0.382 0.181 Pl. tener v tener 5.899 2.001 2.95 0.291 0.130 Pl. cusickii 7.864 3.812 2.10 0.395 0.339 Pl. diffusus 8.272 3.465 2.40 0.397 0.260 Pl. scouleri 7.399 2.594 2.90 0.314 0.196 Pl. cognatus 8.701 3.589 2.42 0.733 0.385 Pl. corymbosus 6.601 2.630 2.50 0.505 0.218 Pl. calandrinoides 9.400 4.044 2.32 0.567 0.403 Pl. albiflorus 11.147 4.024 2.77 1.022 0.351 Pl. scriptus 7.404 3.350 2.20 0.430 0.384 Pl. undulatus 8.482 3.242 2.62 0.402 0.415 Pl. figuratus 6.292 2.524 2.50 0.370 0.208 Pl. humilis 4.849 2.475 2.00 0.517 0.502 Pl. congestus 7.170 3.350 2.14 0.499 0.658 Pl. linifolius 7.441 3.498 2.10 0.353 0.145 Pl. kunthii 6.015 2.660 2.26 0.901 0.354 Pl. mollis 6.640 2.431 2.70 0.429 0.163 Pl. procumbens 10.755 4.819 2.23 0.504 0.477 Pl. collinus v collinus 7.287 4.097 1.78 0.9193 0.4853 Pl. uliginosus 9.069 4.116 2.20 0.521 0.270 Pl. humistratus 7.025 3.629 1.90 0.586 0.379 Pl. acanthocarpus 8.346 3.930 2.12 0.539 0.298 Pl. distantiflorus 8.455 3.658 2.30 0.753 0.410 Pl. glyptocarpus v glyptocarpus 6.617 2.437 2.70 0.240 0.169 Pl. austinae 7.057 2.816 2.50 0.399 0.226 Pl. greenei 7.408 3.316 2.23 0.349 0.206 Pl. strictus 6.131 2.319 2.60 0.362 0.146 Pl. canescens v catilenense 8.812 3.802 2.32 0.518 0.355 Pl. canescens v canescens 8.903 3.756 2.40 0.363 0.204 Pl. arizonicus 6.955 3.284 2.12 0.392 0.356 Pl. uncinatus 5.891 4.734 1.20 0.323 0.330 Pl. verrucosus 5.423 3.961 1.40 0.341 0.408 Pl. tenellus 5.330 3.953 1.30 0.455 0.380 Pl. collinus v fulvescens 7.039 2.656 2.70 0.297 0.161 Pl. pringlei 7.355 3.587 2.05 0.515 0.268 Pl. collinus v gracilis 7.840 2.966 2.64 0.444 0.274 Pl. collinus v californicus 6.355 1.919 3.31 0.325 0.199

Taxa P E P:E stdev P stdev E Pl. collinus v ursinus 7.322 3.766 1.90 0.484 0.313 Pl. infectivus 7.072 4.086 1.70 0.403 0.282 Pl. fulvus 8.180 4.080 2.00 0.380 0.290 Pe. penicillata 6.900 4.108 1.68 0.532 0.463 Pe. dimorpha 7.162 3.442 2.08 0.390 0.314 Pe. platycarpa 8.573 4.281 2.00 0.345 0.254 Pe. linearis 8.779 5.348 1.64 0.628 0.340 Pe. anomala 8.182 6.146 1.30 0.598 0.481 Pe. brachycera 9.330 6.765 1.40 0.748 0.858 H. palmeri v palmeri 7.107 3.290 2.16 0.271 0.267 H. palmeri v arizonica 7.860 5.196 1.50 0.521 0.630 Pe. setosa 6.476 4.438 1.46 0.540 0.385 Pe. pusilla 9.639 6.042 1.60 0.589 0.583 C. flavoculata 9.144 6.447 1.40 0.461 0.332 C. confertiflora 9.368 5.767 1.60 0.475 0.274 C. simulans 7.645 3.587 2.10 0.332 0.236 C. flaccida 6.540 2.668 2.45 0.298 0.239 C. gracilis 5.988 3.669 1.60 0.467 0.311 C. micromeres 6.680 4.349 1.54 0.487 0.466 C. muricata 5.543 1.921 2.90 0.240 0.130 C. maritima 7.189 3.217 2.23 0.511 0.264 C. costata 9.194 3.954 2.30 0.430 0.314 C. racemosa 7.131 3.553 2.01 0.477 0.201 C. circumscissa 6.844 2.838 2.40 0.339 0.205 A. spectabilis 34.590 21.098 1.60 2.990 2.204 A. intermedia 30.637 21.235 1.44 7.637 3.623 A. menziesii 22.182 17.403 1.27 2.683 3.089 A. eastwoodiae 37.916 25.601 1.50 2.723 2.491 A. retrorsa 33.828 22.006 1.50 2.336 1.794 A. furcata 34.240 24.990 1.40 1.866 1.868 A. vernicosa 40.845 23.990 1.70 4.281 3.248 Pl. kingii v harkensii 7.016 3.211 2.20 0.373 0.219 Pl. kingii v kingii 7.240 3.675 2.00 0.412 0.216 Pl .jonesii 8.534 4.989 1.71 0.467 0.253 M. discolor 16.960 11.276 1.50 1.268 1.564 M. laxa 8.836 4.986 1.80 0.972 0.596

Appendix 12. Figure 23. The original Guilliams (2013) molecular phylogeny with bootstrap values at nodes. Scale axis at the bottom of the figure is in millions of years calibrated by ITS mutation rates (Kay et al. 2006).